过表达miR-199-3p/5p诱导胰腺癌细胞BxPC-3、Panc-1铁死亡的作用机制

Mechanism of ferroptosis induced by overexpression of miR-199-3p/5p in pancreatic adenocarcinoma cells BxPC-3 and Panc-1

目的 探讨过表达miR-199-3p/5p诱导胰腺癌细胞BxPC-3和Panc-1铁死亡的作用机制。方法 瞬时转染miR-199-3p/5p模拟物和抑制物至BxPC-3、Panc-1细胞中。qRT-PCR检测细胞中miR-199-3p/5p表达水平;Western blotting检测SCL7A11、BTRC、TFRC蛋白水平。谷胱甘肽(GSH)、Fe^(2+)、脂质过氧化物(LPO)试剂盒检测细胞GSH、Fe^(2+)、LPO水平。免疫共沉淀检测BTRC、TFRC泛素化水平。双荧光素酶报告实验检测miR-199与靶基因的相互作用。结果 转染miR-199-3p/5p模拟物后,胰腺癌BxPC-3、Panc-1细胞株中miR-199-3p/5p、TFRC蛋白水平显著上调;SLC7A11、BTRC蛋白水平显著降低;LPO、Fe^(2+)含量升高,GSH含量降低。过表达BTRC能够显著降低TFRC蛋白水平。miR-199-3p靶向结合SLC7A11,miR-199-5p靶向结合BTRC。结论 过表达miR-199-3p/5p诱导胰腺癌细胞BxPC-3和PANC-1铁死亡,可能与靶向抑制BTRC、SLC7A11促进LPO累积有关。

Aim To investigate the mechanism of ferroptosis induced by overexpression of miR-199-3p/5p in pancreatic adenocarcinoma cells BxPC-3 and Panc-1.Methods Transient transfection of miR-199-3p/5p mimic and inhibitors into BxPC-3 and Panc-1 cells.qRT-PCR was detected the expression levels of miR-199-3p/5p in cells;Western blotting were used to detect SCL7A11,BTRC,TFRC protein expression levels.Glutathione(GSH),Fe^(2+) and lipid peroxide(LPO) kits were used to detect the levels of GSH,Fe^(2+) and LPO.The regulation of ubiquitination of BTRC and TFRC was detected by immunoprecipitation.The interaction between miR-199 and target genes was detected by dual luciferase reporting assay.Results After transfection of miR-199-3p/5p mimic in pancreatic adenocarcinoma cells BxPC-3 and Panc-1,the levels of miR-199-3p/5p and TFRC protein were significantly up-regulated,and the expression levels of SLC7A11 and BTRC proteins decreased significantly;The level of GSH decreased,and the levels of Fe^(2+) and LPO increased significantly.Overexpression of BTRC can significantly reduce TFRC protein level.miR-199-3p binds to SLC7A11,and miR-199-5p binds to BTRC.Conclusion Overexpression of miR-199-3p/5p induces ferroptosis in pancreatic adenocarcinoma cells BxPC-3 and Panc-1,which may be related to the targeted inhibition of BTRC and SLC7A11 to promote LPO accumulation.