siNrf2对万珂诱导甲状腺癌细胞凋亡影响及其机制的探讨

Effects of siNrf2 on apoptosis of thyroid cancer cells induced by bortezomib and its meohanism

目的:明确Nrf2对蛋白酶体抑制剂万珂诱导甲状腺癌细胞凋亡的影响及其作用途径。方法:选取Nrf2高表达的8305C细胞,用微小RNA干扰技术转染细胞,采用实时定量PCR法和蛋白质印迹法分析封闭效果,而细胞内GCLCmRNA的表达、还原型谷胱甘肽(GSH)的含量和细胞凋亡率,分别由实时定量PCR法、比色法和流式细胞仪法测得。并测定siNrf2对万珂诱导其他甲状腺癌细胞凋亡的影响。结果:与对照组相比,siNrf2组在培养液和万珂处理中Nrf2mRNA和蛋白都显著减少,P<0.01;随机序列核酸siRNA和错位型siNrf2差异无统计学意义,P>0.05。与对照组相比,siNrf2能够增加万珂诱导8305C、8505C、KTC1和KTC3的细胞凋亡率,差异均有统计学意义,P<0.01;FRO和KTC2的细胞凋亡率差异无统计学意义,P>0.05。与随机序列核酸siRNA组相比,空白对照组中siNrf2转染组GCLCmRNA及GSH减少,P<0.05;万珂处理组中siNrf2转染组两者减少更显著,P<0.01。GSH和NAC(GSH的前体并能提高GSH的含量)抑制了siNrf2促万珂诱导甲状腺癌细胞凋亡作用。结论:Nrf2抵消万珂诱导甲状腺癌细胞凋亡作用,至少部分是通过GCLC转录激活和随后GSH的生成实现的。

OBJECTIVE： To investigate the role of Nrf2 on the apoptosis of thyroid cancer cells induced by bortezomib.METHODS： 8305C cells,which expressed highest basal Nrf2 in a panel thyroid cancer cells,were selected for the investigation.Small interfering RNA（siRNA） was used to knock down Nrf2.The knockdown efficacy was confirmed by real-time RT-PCR and Western blot.Intracellular GCLC mRNA,glutathione levels and apoptotic cells were measured respectively by real-time RT-PCR,colorimetry and flow cytometry.And the effect of siNrf2 on apoptosis of other thyroid cancer cells induced by bortezomib was measured.RESULTS： siNrf2 significantly reduced Nrf2 mRNA and protein levels in vehicle and bortezomib-treated cells（P〈0.01）,while scramble or mutated siNrf2 had no obvious effects（P〈0.05） when compared with untransfected cells;siNrf2 significantly promoted apoptosis induced by bortezomib in 8305C,8505C,KTC1 and KTC3 cells（P〈0.01）,while siNrf2 had no obvious effects on bortezomib induced apoptosis of FRO and KTC2 cells（P〉0.05）.When compared with scramble siRNA,siNrf2 significantly reduced basal GCLC mRNA and intracellular GSH production（P〈0.05）,siNrf2 had more obvious effects on reduction of GCLC mRNA and intracellular GSH production in bortezomib-treated cells（P〈0.01）.GSH and NAC significantly blocked the enhancing effects of siNrf2 on apoptosis of thyroid cancer cells induced by bortezomib.CONCLUSIONS： Nrf2 suppresses apoptosis of thyroid cancer cells induced by bortezomib,at least in part,via transactivation of GCLC and subsequent generation of GSH.