ENO1缺失诱导胶质母细胞瘤细胞NCOA4介导的铁死亡的机制研究

Mechanism of ENO1 Inducing NCOA4-Mediated Ferroptosis in Glioblastoma Cell

[目的]探究ENO1通过NCOA4对胶质母细胞瘤(GBM)细胞铁死亡的调节机制。[方法]qRT-PCR和Western blot检测GBM组织和正常组织中ENO1的表达。人GBM细胞系U251细胞分为对照组、si-ENO1-NC组、si-ENO1-1组、si-ENO1-2组、si-ENO1+si-NCOA4-NC组、si-ENO1+si-NCOA4组,分别转染siRNA-ENO1-NC、siRNA-ENO1、siRNA-NCOA4-NC或siRNA-NCOA4序列,qRT-PCR和Western blot验证ENO1或NCOA4的表达;Western blot检测铁蛋白重多肽1(FTH1)蛋白表达;MTT法分析U251细胞增殖能力;试剂盒检测U251细胞ROS、MDA、Fe^(2+)含量及线粒体膜电位;透射电镜观察线粒体形态;PI染色检测U251细胞死亡率。[结果]与正常组织相比,GBM组织中ENO1 mRNA和蛋白表达较高(P<0.05)。与对照组和si-ENO1-NC组相比,转染si-ENO1可降低ENO1 mRNA和蛋白表达、FTH1蛋白表达以及24、48、72 h的细胞增殖活力(24 h OD值:0.35±0.05 vs 0.48±0.09,0.35±0.05 vs 0.50±0.08;48 h OD值:0.56±0.07 vs0.98±0.13,0.56±0.07 vs 1.05±0.10;72 h OD值:0.69±0.08 vs 1.35±0.14,0.69±0.08 vs 1.38±0.11)、红/绿荧光比值(3.46±0.79 vs 11.14±1.53,3.46±0.79 vs 10.97±1.82)(P<0.05),增加ROS(7.13±0.69 vs 1.00±0.12,7.13±0.69 vs 0.97±0.16)、MDA(5.61±0.53 vs 1.85±0.26,5.61±0.53 vs 1.83±0.42)、Fe^(2+)含量(6.79±0.78 vs 2.41±0.32,6.79±0.78 vs 2.46±0.47)、细胞死亡率(25.48±3.17 vs 7.31±0.84,25.48±3.17 vs 7.24±1.02)、NCOA4 mRNA和蛋白表达(P均<0.05),si-ENO1的上述作用均可被si-NCOA4逆转。[结论]ENO1缺失可上调NCOA4表达,降低FTH1蛋白水平,进而促进ROS、MDA及铁释放,诱导GBM细胞铁死亡。

[Objective]To explore the effect of α-enolase(ENO1)on nuclear receptor coactivator(NCOA4)-mediated ferroptosis in glioblastoma(GBM)cells and its mechanism.[Methods]The expression of ENO1 in GBM tissues and normal tissues was detected with qRT-PCR and Western blot.Human GBM U251 cells were divided into control group,si-ENO1-NC group,si-ENO1-1 group,si-ENO1-2 group,si-ENO1+si-NCOA4-NC group,and si-ENO1+si-NCOA4 group,the siRNA-ENO1-NC,siRNA-ENO1,siRNA-NCOA4-NC or siRNANCOA4 sequences were transfected in U251 cells,respectively.The expression of ENO1 or NCOA4 was verified by qRT-PCR and Western blot,the expression of FTH1 protein was measured by Western blot,the proliferation ability of U251 cells was analyzed by MTT method,the contents of ROS,MDA,Fe^(2+)and mitochondrial membrane potential in U251 cells were measured by detection kits,the morphology of mitochondria was observed by transmission electron microscope,and the cell death was detected by PI staining.[Results]Compared with normal tissues,the ENO1 mRNA and protein expressions were higher in GBM tissues(P<0.05).Compared with control group and si-ENO1-NC group,the transfection of si-ENO1 reduced ENO1 mRNA and protein expressions,FTH1 protein expression,cell proliferation activity at 24,48 and 72 h(OD24h:0.35±0.05 vs 0.48±0.09,0.35±0.05 vs 0.50±0.08;OD48h:0.56±0.07 vs 0.98±0.13,0.56±0.07 vs 1.05±0.10;OD72h:0.69±0.08 vs 1.35±0.14,0.69±0.08 vs 1.38±0.11),Red/Green fluorescence ratio(3.46±0.79 vs 11.14±1.53,3.46±0.79 vs 10.97±1.82,P<0.05),increase ROS(7.13±0.69 vs 1.00±0.12,7.13±0.69 vs 0.97±0.16),MDA(5.61±0.53 vs 1.85±0.26,5.61±0.53 vs 1.83±0.42),Fe^(2+)contents(6.79±0.78 vs 2.41±0.32,6.79±0.78 vs 2.46±0.47),cell mortality(25.48±3.17 vs 7.31±0.84,25.48±3.17 vs 7.24±1.02),NCOA4 mRNA and protein expressions(all P<0.05),the above effects of si-ENO1 were reversed by si-NCOA4.[Conclusion]The deletion of ENO1 can upregulate the expression of NCOA4,reduce the level of FTH1 protein,and then promote the releases of ROS,MDA and iron,and induce ferroptosis in GBM cells.