| 李维民,刘应刚,石海平,等.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[J].肿瘤学杂志,2023,29(5):394-400. |
| 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 |
| 投稿时间：2022-12-07 |
| DOI：10.11735/j.issn.1671-170X.2023.05.B008 |
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| 中文关键词: 胶质母细胞瘤 α-烯醇化酶 核受体共激活因子4 铁死亡 铁蛋白重多肽1 |
| 英文关键词:glioblastoma alpha-enolase nuclear receptor coactivator 4 ferroptosis ferritin heavy chain 1 |
| 基金项目:四川省医学科研课题项目（S21031） |
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| 中文摘要: |
| 摘 要：［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、Fe2+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 vs 0.98±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）、Fe2+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。 |
| 英文摘要: |
| Abstract： ［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 siRNA-NCOA4 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， Fe2+ 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)， Fe2+ 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 up-regulate 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. |
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