The Effect Of Interfering ?-catenin On Glioma Cell Proliferation And Apoptosis And Its Underlying Mechanism

Part 1:interfering ?-catenin expression represses human glioma cell U251 proliferationObjective:To investigate whether interfering ?-catenin expression inhibits human glioma cell U251 proliferation.Methods:1 Using RNA interfering technology to interfering ?-catenin expression in U251 cells.The U251 cells were devided into 4 trestment groups,and each group was transfected with N.C.siRNA,siRNA1,siRNA2,siRNA3.Then the ?-catenin level was measured by WB and RT-PCR.2 The U251 cells were devided into control group,N.C.siRNA group,and siRNA2 group,and the control group was added transfected regent,simultaneously N.C.siRNA group and siRNA2 group were transfected with N.C.siRNA and siRNA2.Then the expression of some proliferation-related genes(cyclin D1,c-Myc,c-jun)was measured by RT-PCR.3 the viability of migration of U251 were determined by MTT.4 the ability of migration of U251 were determined by wound scratch assay.Results:1 Compared with N.C.siRNA group,transfection with siRNA1 and siRNA2 significantly decreased the expression of ?-catenin in protein and mRNA level in U251 cell,but siRNA3 exhibited no effect on ?-catenin expression.Notably,siRNA2 was most effective among these three pairs of siRNAs.2 Compared with control group,interferng ?-catenin with siRNA2 significantly decreased the cell viability of U251 and simultaneously proliferation-related gene expression(cyclin D1 and c-Myc).3 Interfering ?-catenin with siRNA2 remarkably decreased the distance of scratch between cells,suggesting that interfering ?-catenin expression significantly reduced cell migration of U251.Conclusion:Interfering ?-catenin expression decreases glioma cell U251 proliferation and cell migration.Part 2:interfering ?-catenin expression increase glioma cell U251 apoptosisObjective:To investigate whether interfering ?-catenin expression can increase glioma cell U251 apoptosis.Methods:1 The U251 cells were devided into control group,N.C.siRNA group,and siRNA2 group,and the control group was added transfected regent,simultaneously N.C.siRNA group and siRNA2 group were transfected with N.C.siRNA and siRNA2.2 Flow cytometry was used to detect apoptosis of U251 with or without siRNA2 interfering.3 RT-PCR and WB were used to measured apoptosis-related gene expression.Results:1 The results from flow cytometry showed that interfering ?-catenin expression significantly increased U251 apoptosis compared with control and N.C.siRNA treatment group.2 Interfering ?-catenin expression significantly increased pro-apoptosis gene(p53,caspase3,caspase9,Bax)expression,conversely decreased anti-apoptosis gene Bcl-2 expression.3 The results from WB showed that interfering ?-catenin expression markedly increased cleaved caspase3 levels.Conclusion:Interfering P-catenin expression significantly increase U251 apoptosis.Part 3:the mechanism of interfering ?-catenin expression-mediated U251 proliferation repression and apoptosisObjective:To investigate the mechanism of interfering ?-catenin expression-mediated U251 proliferation repression and apoptosis.Methods:1 The U251 cells were devided into control group,N.C.siRNA group,and siRNA2 group,and the control group was added transfected regent,simultaneously N.C.siRNA group and siRNA2 group were transfected with N.C.siRNA and siRNA2.2 The protein levels of PI3K,p-Akt,JNK,p-JNK,IKKa/?,p-IKKa/?,and nuclear NF-?B in U251 with or writhout siRNA2 treatment were detected by WB.3 Using PDTC to inhibit NF-?B signaling pathway,the nuclear NF-?B levels was measured by WB.Then the cell viability of U251 was measured by MTT and apoptosis-related genes expression were determined by RT-PCR.Results:1 Compared with control and N.C.siRNA group,interfering ?-catenin expression with siRNA2 exhibited no significantly effects on PI3K,p-Akt,JNK,p-JNK protein levels.Intriguingly interfering ?-catenin expression with siRNA2 significantly increased total IKKa/p,p-IKKa/p,and nuclear NF-?B levels.2 Treatment with PDTC significantly inhibited activation of NF-?B mediated by interfering ?-catenin expression.3 PDTC treatment remarkably restored changed apoptosis-related genes expression and decreased cell viability of U251 mediated by interfering ?-catenin expression.Conclusion:Proliferation repression and apoptosis mediated by interfering ?-catenin expression maybe depend on NF-?B signaling pathway,rather than PI3K-Akt and JNK signaling pathway.Part 4:interfering ?-catenin expression inhibits glioma formation in nude mice Objective:The U251 cells transfected with siRNA2 lentiviryus vectors were subcuneously injected to investigate whether interfering P-catenin expression inhibits glioma oncogenesis in nude mice.Methods:1 The U251 cells were devided into control group,N.C.siRNA group,and siRNA2 group,and the control group was added transfected regent,simultaneously N.C.siRNA group and siRNA2 group were transfected with N.C.siRNA and siRNA2 lentiviryus vectors.2 Using WB and RT-PCR to determine P-catenin protein and mRNA levels to guarantee whether siRNA2 vector was successfully transfected into U251.3 The mice were subcutaneously injected U251 with or without siRNA2 vector transfection,and subsequently the survival rate were recorded.4 The tumor tissues were collected to detected apoptosis-related gene expression by RT-PCR.Results:1 The results from WB and RT-PCR showed that siRNA2 vector transfection significantly decreased ?-catenin expression in protein and mRNA levels,suggesting vector transfection is successful.2 The results from tumorigenesis in nude mice showed interfering ?-catenin expression significantly increased the survival rate of nude mice subcutaneously injected U251 with siRNA2 vector transfection.3 Interfering ?-catenin expression significantly increased pro-apoptosis gene(caspase3,Bax)expression,but deceased ant-apoptosis gene Bcl-2 expression.Conclusion:Interfering ?-catenin expression increase survival rate of nude mice subcutaneously injected U251 with siRNA2 vector transfection and increase apoptosis-related gene expression,suggesting interfering ?-catenin expression can inhibt glioma formation.