miR-485-3p调控肝癌细胞铁死亡的分子机制研究

Research on Molecular Mechanism of miR-485-3p Regulation on Ferroptosis in Hepatocellular Carcinoma Cells

目的:探讨miR-485-3p调控肝癌细胞系HepG2铁死亡的分子机制。方法:使用转染试剂transfect-mate分别转染miR-485-3p模拟物或抑制物至肝癌细胞HepG2中并通过qPCR验证转染效果。加入铁死亡诱导剂索拉非尼(SF),通过CCK-8法检测肝癌细胞增殖水平。通过RT-qPCR验证铁死亡诱导剂作用下肝癌细胞内miR-485-3p表达水平。通过脂质过氧化物试剂盒(MDA)、C11-BODIPY、铁离子检测试剂盒、分别检测肝癌细胞内过氧化物、铁离子积累水平。Western blot测定转染miR-485-3p模拟物或抑制物后核受体共激活因子4 (NCOA4)的表达水平。结果:与“NC + SF”组相比,“Mimics + SF”组肝癌细胞活力减弱,“Inhibitor + SF”组肝癌细胞活力显著增强。qPCR结果显示索拉非尼诱导后,肝癌细胞内miR-485-3p表达水平较对照组升高。“Mimics + SF”组细胞内丙二醛(MDA)、铁离子水平较“NC + SF”组显著升高。共聚焦显微镜下观察到 “Mimics + SF”组肝癌细胞内ROS累积显著,“Inhibitor + SF”组相对较低。Western blot结果显示转染miR-485-3p模拟物后肝癌细胞内NCOA4表达增强,而转染抑制物后NCOA4表达显著下降。结论:miR-485-3p可以通过调控NCOA4影响肝癌细胞铁死亡。

Objective: To investigate the molecular mechanism of miR-485-3p on ferroptosis of hepatocellular carcinoma cell line HepG2. Methods: Transfect-Mate was used to transfect miR-485-3p mimics or inhibitors into HepG2 cells, and the transfection effect was verified by qPCR. Adding Sorafenib (SF) as ferroptosis inducer, the proliferation of hepatocellular carcinoma cells was detected by CCK-8 as-say. RT-qPCR was used to verify the expression level of miR-485-3p in hepatocellular carcinoma cells under the effect of ferroptosis inducer. The accumulation levels of superoxide and iron in HCC cells were detected by Lipid Peroxidation MDA Assay Kit (MDA), C11-BODIPY and Iron assay Kit re-spectively. Western blot was used to determine the expression level of nuclear receptor coactivator 4 (NCOA4) after transfection with miR-485-3p mimic or inhibitor. Results: Compared with “NC + SF” group, the viability of hepatocellular carcinoma cells in “Mimics + SF” group was decreased, while that in “Inhibitor + SF” group was significantly increased. qPCR results showed that after sorafenib induction, the expression level of miR-485-3p in hepatocellular carcinoma cells was higher than that in the control group. The levels of malondialdehyde (MDA) and intracellular iron in the “Mimics + SF” group were significantly higher than those of “NC + SF” group. It was observed under confocal microscope that ROS accumulation in hepatocellular carcinoma cells of the “Mimics + SF” group was significant, while the “Inhibitor + SF” group was relatively low. Western blot results showed that the expression of NCOA4 in hepatocellular carcinoma cells was enhanced after transfection with miR-485-3p mimics, while the expression of NCOA4 was significantly decreased after transfection with inhibitor. Conclusion: miR-485-3p affects ferroptosis in hepatocellular carcinoma cells by reg-ulating NCOA4.