[Effects of ATG5 and ATG7 Knockout on Ferroptosis Sensitivity of RPMI-8226 Cells].

Q4 Medicine

中国实验血液学杂志 Pub Date : 2024-10-01 DOI:10.19746/j.cnki.issn.1009-2137.2024.05.022

Jing-Jing Jiang, Chong Chen, Li Nian, Yang Liu, Jin Ma, Cong-Yue Wang, Zhen-Yu Li

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Abstract

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.

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[ATG5 和 ATG7 基因敲除对 RPMI-8226 细胞铁氧化敏感性的影响］

目的研究ATG5和ATG7基因对多发性骨髓瘤（MM）细胞系RPMI-8226细胞自噬敏感性的影响：方法：利用CRISPR/Cas9技术敲除RPMI-8226细胞中的自噬关键基因ATG5和ATG7。方法：利用 CRISPR/Cas9 技术敲除 RPMI-8226 细胞中的自噬关键基因 ATG5 和 ATG7，通过 Western 印迹鉴定基因敲除细胞，并检测自噬相关蛋白 P62 和 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（ROS）水平：结果：Western blot结果证实，RPMI-8226细胞成功敲除了ATG5和ATG7基因。流式细胞术结果表明，RPMI-8226 细胞的活力与不同浓度的 RSL3 的剂量相关（r =-0.969）。用RSL3（10 μmol/L）诱导对照组和基因敲除组细胞的铁变态反应，48小时后基因敲除组的细胞活力均高于对照组（P均<0.001）。敲除ATG5和ATG7基因后，细胞内Fe2+含量较对照组显著下降（均P＜0.01），ROS水平也有所下降（均P＜0.001）：结论：敲除ATG5和ATG7基因可抑制MM细胞的铁突变，LAP通路可能参与了调控。

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