目的：探究雷公藤甲素（TPL）通过miR-34b-5p调控Notch1表达对骨肉瘤U2OS细胞铁死亡影响的机制。方法：常规培养U2OS细胞，将其分为对照组、TPL（10 μmol/L）组、TPL（10 μmol/L）+Fer-1（铁死亡抑制剂，20 μmol/L） 组、miR-NC组、miR34b-5p组、miR-34b-5p+Fer-1（20 μmol/L）组、TPL（10 μmol/L）+anti-miR-34b-5p 组、anti-miR-34b-5p+Fer-1（20 μmol/L）组。qPCR法、CCK-8法、铁离子检测试剂、DHE-荧光探针和WB法分别检测各组U2OS细胞中miR-34b-5p的表达、增殖能力、Fe2+水平、ROS水平以及铁死亡相关蛋白（GPX4、SLC7A11及Notch1蛋白）的表达，双萤光素酶报告基因实验验证miR-34b-5p与Notch1的靶向结合关系。结果： TPL可促进U2OS细胞中miR-34b-5p表达，Fer-1和anti-miR-34b-5p则抑制miR-34b-5p的表达（均P<0.05）。TPL明显抑制U2OS细胞的增殖、GPX4、SLC7A11、Notch1蛋白的表达、增加细胞中Fe2+和ROS的含量，Fer-1可逆转TPL对U2OS细胞的作用（均P<0.05）。过表达miR-34b-5p与TPL对U2OS细胞的作用相似（均P<0.05）。miR-34b-5p可靶向结合Notch1（均P<0.05）。miR-34b-5p抑制剂可明显抑制TPL对U2OS细胞的影响，Fer-1可增强miR-34b-5p抑制剂的作用（均P<0.05）。结论：TPL可抑制U2OS细胞的增殖能力并促进其铁死亡，其作用机制可能与miR-34a-5p靶向调节Notch1表达有关。

Objective: To investigate the mechanism of triptolide (TPL) regulating Notch1 expression on ferroptosis of osteosarcoma U2OS cells via miR-34b-5p. Method: U2OS cells were routinely cultured and divided into the control group , the TPL (10 μmol/L) group, the TPL (10 μmol/L)+Fer-1 (ferroptosis inhibitor, 20 μmol/L) group, the miR-NC, miR-34b-5p, miR-34b-5p+Fer-1 (20 μmol/L) group, the TPL (10 μmol/L)+anti-miR-34b-5p group, and the anti-miR-34b-5p+Fer-1 (20 μmol/L) group. qPCR assay, CCK-8 assay, ferric ion detection reagent, DHE-fluorescent probe, and WB assay were used to detect the expression of miR-34b-5p, the proliferative ability, and the level of Fe2+, ROS levels and the expression of GPX4, SLC7A11 and Notch1 proteins, respectively. Dual luciferase reporter gene assay was used to verify the targeted binding relationship between miR-34b-5p and Notch1. Results: TPL promoted miR-34b-5p expression in U2OS cells, while Fer-1 and anti-miR-34b-5p inhibited miR-34b-5p expression (all P<0.05）. TPL significantly inhibited the proliferation of U2OS cells, the expression of ferroptosis-related proteins (GPX4, SLC7A11, and Notch1 proteins), and increased the cellular Fe2+ and ROS content, and Fer-1 reversed the effect of TPL on U2OS cells (all P<0.05）. Overexpressing miR-34b-5p had similar effects on U2OS cells as TPL (all P<0.05）. miR-34b-5p can be targeted to bind Notch1 (all P<0.05）. miR-34b-5p inhibitor could significantly inhibit the effect of TPL on U2OS cells (all P<0.05）. Fer-1 could enhance the effect of miR-34b-5p inhibitor (all P<0.05）. Conclusion: TPL inhibits the proliferative capacity and promotes ferroptosis of U2OS cells, and its mechanism may be related to the targeted regulation of Notch1 expression by miR-34a-5p.

贵州省科学技术厅黔科合基础（No.ZK[2022]544）