| OBJECTIVE: To investigate effects of oridonin(ORI)on inhibition of U87MG cells and explore the molecular mechanism whether involved the inhibition of UNC45A(Uncoordinated-45 homolog A)/YAP(Yes-associated protein)signaling pathway.METHODS: The expression level of UNC45A in glioma patients was obtained from the American Cancer and Tumor Gene Atlas(Cancer Genome Atlas,TCGA)database and the relationship between UNC45A expression levels and survival time of glioma patients was analyzed.In order to study the effect of UNC45A on YAP signal transduction in U87MG,U87MG were transfected with lentivirus,and established NC-siUNC45A-U87MG and siUNC45A-U87MG,western blot(WB)and quantitative real-time PCR(qPCR)were used to detect the transfection efficiency,and then divided into 3 groups including the U87MG group,NC-siUNC45A-U87MG group and siUNC45A-U87MG group,the growth activity of each cell line was detected by MTT assay,the colony formation was used to detected by cloning experiment,the cell cycle and the apoptosis of each cell line were detected by flow cytometry(FCM),the expression of Ki67 was detected by immunofluorescence(IF),protein expression of caspase3,BAX,Bcl-2,UNC45A,YAP,p-YAP(Ser127)and c-Myc were detected by WB.In order to study the anti-U87MG effect of ORI,U87MG cells were treated with different concentrations of ORI,the migration ability of U87MG was detected by scratch test,the other index and methods were same with the former study.In order to further clarify the effect of oridonin on UNC45A/YAP signal transduction in U87MG,U87MG were transfected with lentivirus,and established NC-overUNC45A-U87MG and overUNC45A-U87MG,WB and qPCR were used to detect the transfection efficiency,and then divided into 5groups including U87MG group,ORI+ U87MG group,NC-overUNC45A-U87MG group,overUNC45A-U87MG group,and ORI+ overUNC45A-U87MG group.The index and methods were same with the former study and the relationship between the effect of oridonin against U87MG and UNC45A/YAP pathway was discussed.RESULTS:1.The role of UNC45A/YAP signaling pathway in U87MG proliferationFrom TCGA database,we found glioma patients with high expression of UNC45A have longer survival than glioma patients with low expression of UNC45A.In the cellular experiments,compared with the control group,siUNC45A-U87MG cells had weaker growth activity(P< 0.01),less colony formation(P< 0.01),the proportion of G1 phase cells was increased(P< 0.01)the expression of Ki67(P< 0.01)was decreased,the protein expression of caspase-3,BAX and p-YAP(Ser127)were increased(P<0.01)and the protein expression of Bcl-2,YAP and c-Myc were decreased significantly(P< 0.01).2.The anti-U87MG effect of oridoninCompared with the control group,ORI showed the effect of inhibition the growth of U87MG in a time and dose dependent manner(P< 0.01),as well as the colony formation and migration of U87MG were decreased(P<0.01),the proportion of G2/M phase cells was increased(P< 0.01),and the apoptotic rate was increased significantly(P< 0.01);the protein expression of caspase3,BAX,p-YAP(Ser127)were increased(P< 0.01)and the protein expression of Bcl-2,UNC45A,YAP,c-Myc were decreased significantly(P< 0.01).3.Effect of oridonin on U87MG and its relationship with UNC45A/YAP signaling pathwayCompared with the control group,overUNC45A-U87MG cell growth activity(P< 0.01)and clone formation increased significantly(P< 0.01);there was no significant difference in the proportion of cells in each cycle and(P> 0.05)and the expression of Ki67(P> 0.05),and the expression of Bcl-2,c-Myc,YAP and p-YAP were increased significantly(P< 0.05).Compared with the control group,the ORI treated U87MG group showed the significantly decrease of viability(P< 0.01),the colony formation(P<0.01),the proportion of cells in G2/M phase(P< 0.01)and the expression of Ki67,Bcl-2,YAP and c-Myc(P< 0.01)as well as increased significantly,the expressions of BAX,caspase-3 and p-YAP were decreased significantly(P< 0.01).Compared with overUNC45A-U87MG group,overUNC45A-U87MG cells treated with 8μM ORI showed the significant decrease in cell viability(P< 0.01)and colony formation(P< 0.01),but no significant difference in the proportion of cells in each cycle(P> 0.05),the expression of Ki67,Bcl-2,YAP and c-Myc were decreased(P< 0.01)and the expressions of BAX,caspase-3 and p-YAP were increased significantly(P< 0.01).CONCLUSION:1)Knockdown UNC45A can inhibit the expression of YAP and then show the anti-glioblastoma effect.2)Oridonin can inhibited proliferation and induced apoptosis of U87MG in a dose-dependent manner.3)The mechanism of oridonin inhibiting proliferation and promoting apoptosis in U87MG involves the inhibition of UNC45A/YAP signaling pathway. |
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