Objective: To investigate the effects of ATG5 and ATG7 genes on the sensitivity of multiple myeloma (MM) cell line RPMI-8226 cells to ferroptosis.
Methods: CRISPR/Cas9 technology was used to knock out the autophagy key genes ATG5 and ATG7 in RPMI-8226 cells. Western blot was used to identify gene knockout cells, and detect the expression changes of autophagy-related proteins P62 and LC3B. Flow cytometry was used to detect the change of sensitivity of gene knockout cells to RSL3. The content of intracellular ferrous ions and reactive oxygen species (ROS) level in gene knockout cells were detected.
Results: Western blot result confirmed that ATG5 and ATG7 genes were knocked out successfully in RPMI-8226 cells. The result of flow cytometry showed that the cell viability of RPMI-8226 cells was dose-dependent on different concentrations of RSL3 (r =-0.969). RSL3 (10 μmol/L) was used to induce ferroptosis in cells of control group and gene knockout groups, then the cell viability in gene knockout groups were both higher than control group after 48 hours (both P < 0.001). After knocking out the ATG5 and ATG7 genes, the content of intracellular Fe2+ decreased significantly compared with control group (both P < 0.01), and the ROS level also decreased (both P < 0.001).
Conclusion: Knockout of ATG5 and ATG7 genes can inhibit the ferroptosis of MM cells, and LAP pathway may be involved in the regulation.
题目: 敲除ATG5、ATG7基因对RPMI-8226细胞铁死亡敏感性的影响.
目的: 探讨ATG5、ATG7基因对诱导多发性骨髓瘤细胞系RPMI-8226细胞发生铁死亡的敏感性的影响。.
方法: 用CRISPR/Cas9技术敲除RPMI-8226细胞株中自噬关键基因ATG5和ATG7；Western blot法鉴定基因敲除细胞并检测自噬相关蛋白P62、LC3B的表达变化；流式细胞术检测基因敲除细胞对RSL3敏感性的变化；检测基因敲除细胞内亚铁离子含量和活性氧水平。.
结果: Western blot检测结果证实RPMI-8226细胞中ATG5、ATG7基因被成功敲除。流式细胞术检测结果显示，RPMI-8226细胞存活率与不同浓度RSL3呈剂量依赖关系（r =-0.969）；用10 μmol/L的RSL3诱导对照组和基因敲除组细胞发生铁死亡，48 h后基因敲除组细胞存活率显著高于对照组（均P < 0.001）。ATG5、ATG7基因敲除后，细胞内Fe2+的含量较对照组明显下降（均P < 0.01），活性氧水平亦明显下降（均P < 0.001）。.
结论: 敲除ATG5、ATG7基因可以抑制多发性骨髓瘤细胞发生铁死亡，LAP途径可能参与其调控。.
Keywords:
ferroptosis;