| 王 微,夏鑫杭,杨海华,等.磷酸酶PHLPP2抑制GPX4活性增强非小细胞肺癌铁死亡诱导剂RSL3敏感性的作用分析[J].肿瘤学杂志,2022,28(6):459-464. |
| 磷酸酶PHLPP2抑制GPX4活性增强非小细胞肺癌铁死亡诱导剂RSL3敏感性的作用分析 |
| Phosphatase PHLPP2 Enhances Ferroptosis Inducer RSL3 Sensitivity by Inhibiting GPX4 Activity in Non-small Cell Lung Cancer |
| 投稿时间：2022-01-11 |
| DOI：10.11735/j.issn.1671-170X.2022.06.B004 |
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| 中文关键词: 非小细胞肺癌 铁死亡 PHLPP2 |
| 英文关键词:non-small cell lung cancer ferroptosis PHLPP2 |
| 基金项目:浙江省基础公益技术研究计划（LGF21H160027）；台州市科技计划项目（20ywa09） |
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| 中文摘要: |
| 摘 要：［目的］ 探讨磷酸酶PHLPP2在铁死亡诱导剂RSL3诱导非小细胞肺癌细胞铁死亡发生中的作用及机制。［方法］ 体外培养人肺癌细胞株，GPX4抑制剂RSL3处理后MTT法检测RSL3抑制非小细胞肺癌细胞增殖作用，实时荧光定量PCR检测mRNA水平，Western blot法检测蛋白表达。流式细胞术检测脂质ROS水平，分别采用逆转录病毒/腺病毒感染上调/下调PHLPP2表达，两组间比较采用独立样本t检验。［结果］ RSL3有效抑制非小细胞肺癌细胞的活力，RSL3处理后HCC827、H1975、H1650、A549和H1299细胞的增殖率分别为15.22%±2.35%、 25.05%±5.92%、 4.40%±1.61%、41.48%±10.01%和13.17%±2.54%。 PHLPP2表达水平最高的H1650细胞对RSL3抑制增殖作用最显著，RSL3抑制增殖作用与 PHLPP2表达水平呈正比。上调PHLPP2表达可以增加RSL3对A549细胞增殖抑制作用，并增加铁死亡关键标志脂质ROS的累积，A549-vector和A549-PHLPP2组的脂质ROS水平分别为 4.34%±0.39%和15.36%±0.80%（P<0.001）；相反，下调PHLPP2表达H1650细胞中，RSL3对H1650细胞增殖抑制作用减弱，减少了其诱导的脂质ROS的累积，H1650-shControl和H1650-shPHLPP2脂质ROS水平分别为14.76%±1.22%和4.89%±1.81%（P=0.0 015）。公共数据库挖掘分析PHLPP2与铁死亡关键蛋白GPX4、SLC7A11和ASCL4的相关性，发现PHLPP2与GPX4表达呈负相关（r=-0.336，P<0.001）。Western blot进一步验证了上调PHLPP2表达可增强RSL3对GPX4蛋白的抑制作用。［结论］ PHLPP2促进GPX4抑制剂RLS3诱导铁死亡发生，其机制主要通过协同抑制GPX4活性。 |
| 英文摘要: |
| Abstract： ［Objective］ To explore the effect and mechanism of phosphatase PHLPP2 on ferroptosis inducers(FINs) RSL3 sensitivity in non-small cell lung cancer(NSCLC) cells. ［Methods］ Human NSCLC cell lines A549， H1650， HCC827， H1975 and H1299 were treated with GPX4 inhibitor RSL3， the cell proliferation was assessed by MTT assay in NSCLC cell lines. Real-time fluorescence quantitative PCR(qRT-PCR) and Western blot were used to analyze PHLPP2 expression. Lipid reactive oxygen species(ROS) was assessed by flow cytometry using fluorescent probes C11-BODIPY. Retrovirus/adenovirus transfection was used to up-regulate and down-regulate the expression of PHLPP2 in A549 and H1650 cells， respectively. Two-tailed unpaired Student’s t-test was used for comparison between the two groups. ［Results］ RSL3 effectively inhibited the viability of NSCLC cells. After RSL3 treatment， the proliferation rates of HCC827， H1975， H1650， A549 and H1299 cells were 15.22%±2.35%， 25.05%±5.92%， 4.40%±1.61%， 41.48%±10.01% and 13.17%±2.54%， respectively. Overexpression of PHLPP2 promoted cell death and increased lipid ROS production induced by RSL3 in A549 cells， the lipid ROS production in the A549-vector and A549-PHLPP2 groups were 4.34%±0.39% and 15.36%±0.80%， respectively（P<0.001）. On the contrary， in H1650 cells with down-regulated PHLPP2 expression， RSL3 attenuated the proliferation inhibition of H1650 cells and reduced the accumulation of lipid ROS induced by RSL3. The levels of lipid ROS of H1650-shControl and H1650-shPHLPP2 were 14.76%±1.22% and 4.89%±1.81%， respectively(P=0.0 015). Public database mining analysis showed the correlation between PHLPP2 and iron death key proteins GPX4， SLC7A11 and ASCL4. It was found that PHLPP2 was negatively correlated with the expression of GPX4(r=-0.336， P<0.001). Western blot further verified that up-regulating the expression of PHLPP2 enhanced the inhibitory effect of RSL3 on GPX4 protein. ［Conclusion］ PHLPP2 may promote ferroptosis induced by GPX4 inhibitor RLS3 through synergistic inhibition of GPX4 activity. |
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