Zhongguo shi yan xue ye xue za zhi · 2024
[Effects of ATG5 and ATG7 Knockout on Ferroptosis Sensitivity of RPMI-8226 Cells].
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Jiang Jing-Jing, Chen Chong, Nian Li, Liu Yang, Ma Jin, Wang Cong-Yue, Li Zhen-Yu
Аннотация
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.
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.