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目的 探讨溶血磷脂酰胆碱酰基转移酶3(LPCAT3)在爱拉斯汀(Erastin)诱导急性髓系白血病(AML)细胞铁死亡中的作用及其相关分子调控机制。方法 四唑盐(MTS)法检测不同AML细胞对铁死亡经典诱导剂Erastin的敏感性，实时荧光定量PCR(qPCR)检测其LPCAT3 mRNA的基础表达水平，分析二者关联性。构建慢病毒介导的LPCAT3过表达AML细胞株(OE组)及阴性对照株(NC组)。在Erastin干预后，采用MTS、流式细胞术及微量法分别检测细胞活力、脂质活性氧(ROS)和丙二醛(MDA)。qPCR和Western blot检测未折叠蛋白反应(UPR)经典通路信号分子(PERK、ATF4、GRP78等)的表达水平。采用UPR抑制剂4-苯基丁酸(4-PBA)联合干预后检测上述铁死亡相关指标，分析调控关系。结果 4种不同AML细胞对铁死亡敏感性不同，其中K562细胞相对不敏感，4种AML细胞对Erastin的IC50与LPCAT3表达水平呈负相关(r=-0.919,P<0.001)。Erastin干预后，与NC组相比，OE组K562细胞的细胞活力被Erastin抑制(P<0.001),脂质ROS与MDA水平增加(P<0.001);qPCR、Western blot检测结果显示，与NC组相比，OE组中UPR经典通路分子PERK、ATF4、GRP78 mRNA和蛋白表达均升高(P<0.01);通过4-PBA抑制UPR通路后，与未抑制状态相比，K562细胞活力下降(P<0.01),脂质ROS与MDA水平升高(P<0.01)。结论 过表达LPCAT3可促进K562细胞铁死亡，且其激活的UPR经典通路PERK/ATF负向调控该过程。
Abstract：Objective To explore the role of lysophosphatidylcholine acyltransferase 3(LPCAT3) in Erastin-induced ferroptosis of acute myeloid leukemia(AML) cells and its related molecular regulatory mechanisms. Methods Tetrazolium salt(MTS) method was used to detect the sensitivity of different AML cells to the classic ferroptosis inducer Erastin, real time quantitative polymerase chain reaction(qPCR) was used to detect the basal expression level of LPCAT3 mRNA, and the correlation between them was analyzed. Lentivirus-mediated LPCAT3 overexpression AML cell lines(OE group) and negative control lines(NC group) were constructed. After Erastin intervention, MTS, flow cytometry, and micromethods were used to detect cell viability, lipid reactive oxygen species(ROS), and Malondialdehyde(MDA), respectively. qPCR and Western blot were used to detect unfolded protein response(UPR) classic pathway signaling molecules(PERK, ATF4, GRP78, etc.) expression levels. The above ferroptosis-related indicators were detected after combined intervention with the UPR inhibitor 4-phenylbutyric acid(4-PBA), and the regulatory relationship was analyzed. Results Four different types of AML cells had different sensitivities to ferroptosis, among which K562 cells were relatively insensitive. The IC50 of the four types of AML cells to Erastin was negatively correlated with the expression level of LPCAT3(r=-0.919,P<0.001). After Erastin intervention, the cell viability of K562 cells in the OE group was significantly inhibited by Erastin compared with the NC group(P<0.001), and the levels of lipid ROS and MDA increased(P<0.001). The results of qPCR and Western blot showed that, compared with the NC group, the mRNA and protein expression of UPR classic pathway molecules PERK, ATF4, and GRP78 mRNA and protein increased in the OE group(P<0.01). After inhibiting the UPR pathway by 4-PBA, the viability of K562 cells decreased(P<0.01), and lipid ROS and MDA levels increased(P<0.01) compared with the uninhibited state. Conclusion Overexpression of LPCAT3 can promote ferroptosis in K562 cells, and this process is negatively regulated by the classical UPR pathway PERK/ATF.
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基本信息:
DOI：10.19405/j.cnki.issn1000-1492.2025.01.003
中图分类号:R733.71
引用信息:
[1]江贤栋,黄莹莹,洪小颖等.爱拉斯汀对急性髓系白血病细胞铁死亡的影响及其相关作用机制[J].安徽医科大学学报,2025,60(01):15-21.DOI:10.19405/j.cnki.issn1000-1492.2025.01.003.
基金信息:
福建省自然科学基金面上项目(编号：2021J01815); 国家自然科学基金面上项目(编号：82072355)~~